Detecting photons and producing electronic images from a scanned field of view has been performed to produce electronic outputs representing the field of view of an instrument, such as a laser scanning confocal microscope. In this regard, the term “photon” means a unit of electromagnetic energy irrespective of its position in the spectrum, e.g. visible or invisible radiation. In quantum physics, a photon is characterized as a particle or a wave. The nature of the present invention and the manner of its use are not dependent on whether or not the photon is a particle or a wave.
In one prior art optical detection technique, photons are directed by a confocal imager in the confocal microscope to be sensed by a detector. A confocal imager comprises a point source of light that illuminates a spot within a sample. In order to illuminate an entire sample with a spot, the light source is scanned across a sample by a beam steering device using scanners that are well known in the art. An illuminated spot is then imaged onto a detector through a pinhole, or “point” aperture. Detectors comprise, for example, avalanche photodiode arrays or photomultiplier tubes.
The light source, the illuminated spot, and the detector have the same foci; they are placed in conjugate focal-planes. They are therefore “confocal” to each other.
The diameter of the detector aperture is preferably matched to the illuminated spot through the intermediate optics. Because a small spot is illuminated and then detected through a small aperture, only the plane in focus within the specimen is imaged on to the detector. The detector produces output pulses indicative of detected photons.
The detector output pulses are processed to provide information such as time-correlated photon-counting histograms and image generation in conventional laser scanning. In conventional imaging systems, however, photons obtained over each of a number of successive, selected equal time periods defined by a pixel clock are used to generate an intensity value assigned to each pixel (two-dimensional area of a portion of an image). Photon counts are binned, that is, accumulated as a group, during each sampling period corresponding to a pixel location of an image display. In this manner, a computer builds up an entire image one pixel at a time to produce an entire two-dimensional image often made up of thousands or multiple millions of pixels. In three-dimensional imaging, successive two-dimensional layers of a sample are scanned, and the computer builds up an image comprising voxels.
In producing a conventional image, a scan rate is selected. As scan rate increases, fewer photons per pixel per scan are accumulated, and intensity of pixels and signal-to-noise ratio therefore decrease. As a result, prior art pixel-based imaging systems face constraints in scan rate with regard to the quality of output signal to be produced. Physical and mechanical constraints, such as the rate at which a scanner can move, are also present. In addition, the number of photon counts in a sample affects other parameters relating to intensity. These parameters include signal-to-noise ratio.
As a result, pixel based scanning typically allows for reduced flexibility in experiment design. Resolution of the location of each photon is limited to the dimensions of a pixel or voxel as applicable. The amount of excitation illumination required for output data to reach convergence of features of sensed images is proportional to the number of photons that must be produced to provide data sufficient to reach this convergence. When pixels are of smaller dimension and therefore provide fewer photons per scan, samples often must be subjected to excitation radiation a larger number of times than if the pixels were larger.
The requirement for greater illumination has functional drawbacks. In the subset of applications using fluorescent samples, many molecules under test can fluoresce only a limited number of times. At some point, response to excitation radiation ceases, and an effect known as photo-bleaching occurs. Over illumination also presents another drawback. Where measurements are made in vivo, emission of a photon from tissue causes free radicals, which can damage cells. Therefore, over-illumination of tissue can result in photo-toxicity.
A limitation of typical prior art techniques is that they are optically based. Optically based techniques have an inherent limit of resolution known as a diffraction limit, which may be ˜0.6λ, where λ is the wavelength of the illuminating light. The resolving power of a lens is ultimately limited by diffraction effects. The lens' aperture is a “hole” that is analogous to a two-dimensional version of the single-slit experiment. Light passing through it interferes with itself, creating a ring-shaped diffraction pattern known as the Airy pattern, that blurs the image. An empirical diffraction limit is given by the Rayleigh criterion:
      sin    ⁢                  ⁢    θ    =      1.22    ⁢          λ      D      where θ is the angular resolution, λ is the wavelength of light, and D is the diameter of the lens. A wave does not have to pass through an aperture to diffract. For example, a beam of light of a finite size passing through a lens also undergoes diffraction and spreads in diameter. This effect limits the minimum size d of spot of light formed at the focus of a lens, known as the diffraction limit:
      d    =          2.44      ⁢      λ      ⁢              f        a              ,where λ is the wavelength of the light, f is the focal length of the lens, and a is the diameter of the beam of light, or (if the beam is filling the lens) the diameter of the lens. Optical techniques do not afford the opportunity to obtain resolution beyond the diffraction limit.